Owing to the recent setbacks in vaccine and microbicide trails, novel strategies to slow the HIV-pandemic are desperately needed. Development of a small molecule that blocked a host process required for HIV-1 replication could provide such a strategy. Like all retroviruses, HIV-1 requires host proteins to complete its life cycle, and these intracellular host molecules represent an undeveloped pool of novel anti-HIV therapeutics. The goal of this application is to use functional proteomics to identify host proteins that are dispensable to the host but essential for viral replication. A generic proteomic screen would likely identify numerous host proteins induced by HIV-infection, most of which would be poor therapeutic candidates. A functional proteomics screen, which queries a subset of proteins with strong therapeutic potential, would greatly simplify the search for host targets; the host purinome has this potential. The purinome comprises any protein that binds purine- containing molecules (e.g. ATP, NADH), and it includes heat shock proteins, dehydrogenases, and protein kinases. Inhibition of purinome proteins forms the basis of many current therapies, including those targeting cancer, hypertension, and bacterial infections. The central hypothesis of this application states that inhibition of purinome proteins, which are induced or regulated by HIV-infection, will block HIV-1 replication. Our research team has designed a protein affinity media that captures the entire purinome through its purine-binding pocket. The proposed research will proceed in four broad steps: use SILAC (stable isotope labeling with amino acids in cell culture) to quantify purinome proteins induced by HIV-1 infection of THP1 and Jurkat cells (target identification); determine which purinome proteins are essential for HIV-1 replication (target validation); identify drugs that selectively compete validated targets from the purinome-binding resin (drug discovery); confirm that these compounds block HIV-replication in primary cells (drug validation). During this process, we will also identify host proteins that interact with HIV-1, and in the event that a viable purinome target and/or drug is not identified, viral biology will be advanced by a greater understanding of how HIV-1 interacts with its host. The proposed research uses innovative proteomics technology (SILAC) and an unconventional, yet validated, approach. First, conventional anti-HIV therapeutics inhibit virally-encoded proteins, which makes them more susceptible to the development of resistance. By targeting a host enzyme that is required for HIV-1 replication, it is likely that the virus will have to undergo more radical changes to offset the changes in the host milieu. Second, conventional drug discovery uses a reductionist approach to separately identify a target and a drug, while our approach combines the target/drug identification process, which makes discovery more efficient. At the completion of this project, our combination of experience and technology will enable us to characterize host purine-binding proteins that are essential for HIV-1 replication and identify several lead compounds that will inhibit host proteins necessary for HIV-1 replication.